Abstract

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.

Nodulation efficiencies of the wild-type strain and the exoR95 and exoS96 mutants. The average numbers of nodules per plant on alfalfa plants inoculated with Rm1021 and the exoR395 and exoS396 mutants were plotted based on the size of inoculum. The numbers of nodules were further divided into pink nodules (black bars) and white nodules (white bars).

Root hair invasion efficiencies of the wild type and its succinoglycan-overproducing mutants. (A) Average numbers of total invasion events per plant on plants inoculated with wild-type strain Rm1021 and exoY210, exoR95, exoR395exoY210, exoS96, and exoS396exoY210 mutants. (B) Overall efficiencies of root hair invasion of the same set of strains.

The exoR95 and exoS96 mutations blocked flagellum biosynthesis. The wild-type strain and the succinoglycan-deficient mutant exoY210 produced peritrichous flagella. The exoR95, exoS96, exoR395exoY210, and exoS396exoY210 mutants had no flagella.

The exoR95 and exoS96 mutations suppressed expression of the flaA gene. The primers for both the rpsF and visR genes were mixed with total RNA from the wild type or the exoR95 and exoS96 mutants for RT-PCR. (A) The products of the RT-PCRs were resolved on an agarose gel and used to examine the expression of visR and rpsF. (B and C) Expression of the visN and rpsF genes (B) and of the flaA and rpsF genes (C) was similarly examined.